Abstract Studies on patients with inherited defects in cobalamin or B12 metabolism have led to the discovery of eight genes dedicated to the processing, delivery and utilization of this essential cofactor in humans. The gene products are a medley of enzymes that catalyze novel chemical reactions needed for tailoring the cofactor into its active form, G-protein chaperones that employ nucleotide-powered switches to gate and edit cofactor docking and proteins of as yet unknown function. Biochemical studies on B12 trafficking in our laboratory have started providing insights into an elaborately orchestrated intercompartmental escort service for reaching a rare and reactive cofactor to its target enzymes, methionine synthase and methylmalonyl-CoA mutase. In the next cycle, we will address major gaps in our understanding of B12 trafficking by addressing the following specific aims: (i) elucidate the cytoplasmic pathway that uses two newly discovered proteins, CblC and CblD, and converts incoming alkyl- and cyano-cobalamins into a common intermediate that can be partitioned into synthesis of the active cofactor forms, methylcobalamin and 5'-deoxyadenosylcobalamin, (ii) elucidate the mitochondrial 5'- deoxyadenosylcobalamin branch of the pathway in which adenosyltransferase synthesizes and delivers 5'-deoxyadenosylcobalamin to methylmalonyl-CoA mutase in a process that involves a G-protein sentry. The molecular mechansims for cofactor transfer to the mutase and the bidirectional signaling between the mutase and its G-protein editor will be investigated. We will use a range of spectroscopic, cell biological and structural approaches to unravel the chemical and regulatory strategies that are deployed for intracellular handling of B12 and characterize patient mutations and fibroblasts to elucidate how these steps are corrupted, particularly in the late-onset forms of the disease. The impact of the proposed studies is both fundamental (i.e. gaining insights into the operation of the B12 trafficking pathway that are likely to be relevant for other organic cofactors) and medical (i.e. informing therapeutic options for circumventing blockades and/or exploiting the activities of the trafficking proteins for delivery of cargo (e.g. nitric oxide for biomedical applications) using B12 as a vehicle.